Project Summary/Abstract This application seeks to define new physiological functions for the plasma procarboxypeptidase B, Thrombin- Activatable Fibrinolysis Inhibitor (TAFI). Activated TAFI (TAFIa) is a multifunctional carboxypeptidase with various governing functions in the regulation of fibrinolytic, complement, and inflammatory pathways. Defective activation of TAFI in hemophilia plasma in vitro has been recognized long ago, but we have only recently been able to demonstrate that TAFI activation in hemophilia mice in vivo is also defective. Thus, hemophilia, which is a genetic deficiency of coagulation factor VIII or IX, provides a clinically relevant condition to improve our understanding of the (patho)physiological consequences of TAFI deficiency, since a genetic deficiency of TAFI in humans has not yet been found. Bleeding in patients with hemophilia is characterized by a striking susceptibility for repeated and perpetuated intra-articular bleeding in weight-bearing joints that progresses to debilitating hemophilic arthropathy. The majority of the world?s hemophilia population does not have access to a rigorous supply of clotting factor products for prophylactic treatment and as a result hemophilic arthropathy is and will remain highly prevalent in the adult hemophilia population for many generations to come. Furthermore, initiating clotting factor prophylaxis later in life does not diminish existing hemophilic arthropathy, indicating that new knowledge for mechanisms of hemophilic joint disease (HJD), that may lead to additional therapeutic options for patients with hemophilia and arthropathy, are urgently needed. Arthropathic joints in patients with hemophilia are characterized by pronounced vascular abnormalities due to excessive vascular remodeling. Our work has identified that the defective activation of TAFI drives the vascular abnormalities associated with HJD in mice after joint bleeding. Furthermore, the vascular changes in the joint after bleeding due to the loss of TAFI function are not explained by a loss of TAFI?s antifibrinolytic activity, suggesting that other mechanisms and substrates for TAFI are involved. The proposed studies will test a comprehensive conceptual model aimed at obtaining basic and translational knowledge for the benefit of patients with hemophilia and arthropathy. Our novel hypotheses focus on the loss of attenuation of vascular endothelial growth factor A (VEGF-A) and stromal cell-derived factor- 1? (SDF-1?/CXCL12)) activity by TAFIa as driving forces behind the development of vascular abnormalities in HJD. Three Specific Aims will guide our experimentation. New knowledge as to how a loss of TAFI function contributes to HJD will be the focus of Aim 1. Studies of Aim 2 will test the ability of TAFIa to attenuate VEGF-A activity and how this relates to HJD. Aim 3 is focused on the regulation of SDF-1? activity by TAFIa and the extent to which targeting SDF-1? signaling receptors can prevent and reverse HJD. A highly significant premise is that basic knowledge about the mechanisms responsible for the vascular abnormalities in HJD can be translated to vascular treatments for patients with hemophilic arthropathy, including engineered TAFI variants (Aim 1), antibodies (Aim 2), or small molecules (Aim 3).